Name: targeted in situ mutagenesis of histone genes in budding yeast
Uploaded: 2017-01-26T15:00:00.000+00:00
Duration: 8 min 48 s
Description: Watch this Scientific Journal Video about Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast at JoVE.com

We thank Reine Protacio for helpful comments during the preparation of this manuscript. We express our gratitude to the National Science Foundation (grants nos. 1243680 and 1613754) and the Hendrix College Odyssey Program for funding support.

<ol>
	<li>Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F., Richmond, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+nucleosome+core+particle+at+2.8+A+resolution.">Crystal structure of the nucleosome core particle at 2.8 A resolution.</a> Nature. 389 (6648), 251-260 (1997).</li><li>Campos, E. I., Reinberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histones:+annotating+chromatin.">Histones: annotating chromatin.</a> Annu Rev Genet. 43, 559-599 (2009).</li><li>Rando, O. J., Winston, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+and+transcription+in+yeast.">Chromatin and transcription in yeast.</a> Genetics. 190 (2), 351-387 (2012).</li><li>Duina, A. A., Miller, M. E., Keeney, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Budding+yeast+for+budding+geneticists:+a+primer+on+the+Saccharomyces+cerevisiae+model+system.">Budding yeast for budding geneticists: a primer on the Saccharomyces cerevisiae model system.</a> Genetics. 197 (1), 33-48 (2014).</li><li>Storici, F., Resnick, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delitto+perfetto+targeted+mutagenesis+in+yeast+with+oligonucleotides.">Delitto perfetto targeted mutagenesis in yeast with oligonucleotides.</a> Genet Eng (N Y). 25, 189-207 (2003).</li><li>Gray, M., Kupiec, M., Honigberg, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+genomic+(SSG)+and+random+domain-localized+(RDL)+mutagenesis+in+yeast.">Site-specific genomic (SSG) and random domain-localized (RDL) mutagenesis in yeast.</a> BMC Biotechnol. 4, 7 (2004).</li><li>Erdeniz, N., Mortensen, U. H., Rothstein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning-free+PCR-based+allele+replacement+methods.">Cloning-free PCR-based allele replacement methods.</a> Genome Res. 7 (12), 1174-1183 (1997).</li><li>Brachmann, C. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designer+deletion+strains+derived+from+Saccharomyces+cerevisiae+S288C:+a+useful+set+of+strains+and+plasmids+for+PCR-mediated+gene+disruption+and+other+applications.">Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications.</a> Yeast. 14 (2), 115-132 (1998).</li><li>Lundblad, V., Hartzog, G., Moqtaderi, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulation+of+cloned+yeast+DNA.">Manipulation of cloned yeast DNA.</a> Curr Protoc Mol Biol. Chapter 13, (2001).</li><li>Hoffman, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+yeast+DNA.">Preparation of yeast DNA.</a> Curr Protoc Mol Biol. Chapter 13, (2001).</li><li>Treco, D. A., Lundblad, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+yeast+media.">Preparation of yeast media.</a> Curr Protoc Mol Biol. Chapter 13, (2001).</li><li>Lederberg, J., Lederberg, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replica+plating+and+indirect+selection+of+bacterial+mutants.">Replica plating and indirect selection of bacterial mutants.</a> J Bacteriol. 63 (3), 399-406 (1952).</li><li>Sikorski, R. S., Hieter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+system+of+shuttle+vectors+and+yeast+host+strains+designed+for+efficient+manipulation+of+DNA+in+Saccharomyces+cerevisiae.">A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae.</a> Genetics. 122 (1), 19-27 (1989).</li><li>Johnson, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+mutational+analysis+of+a+histone+H3+residue+in+budding+yeast+provides+insights+into+chromatin+dynamics.">A systematic mutational analysis of a histone H3 residue in budding yeast provides insights into chromatin dynamics.</a> G3 (Bethesda). 5 (5), 741-749 (2015).</li><li>DiCarlo, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+engineering+in+Saccharomyces+cerevisiae+using+CRISPR-Cas+systems.">Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems.</a> Nucleic Acids Res. 41 (7), 4336-4343 (2013).</li><li>Cross, S. L., Smith, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+structure+and+cell+cycle+expression+of+mRNAs+encoded+by+two+histone+H3-H4+loci+in+Saccharomyces+cerevisiae.">Comparison of the structure and cell cycle expression of mRNAs encoded by two histone H3-H4 loci in Saccharomyces cerevisiae.</a> Mol Cell Biol. 8 (2), 945-954 (1988).</li><li>Libuda, D. E., Winston, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplification+of+histone+genes+by+circular+chromosome+formation+in+Saccharomyces+cerevisiae.">Amplification of histone genes by circular chromosome formation in Saccharomyces cerevisiae.</a> Nature. 443 (7114), 1003-1007 (2006).</li></ol>

The authors declare that they have no competing financial interests.

L&#39;elevata omologia di sequenza tra due geni non alleliche che codificano ciascuno dei quattro proteine essenziali istoniche delle cellule S. cerevisiae aploidi possa rappresentare sfida per investigatori vogliano indirizzare specificamente uno dei due geni per mutagenesi. In precedenza descritto lievito metodologie di mutagenesi, compreso il Delitto Perfetto, genomica mutagenesi sito-specifica (SSG), e PCR-based metodi pezzi allele-free clonazione 5, <sup class="x...

I quattro proteine ​​di base dell&#39;istone H2A, H2B, H3, H4 e giocano un ruolo centrale nella compattazione, l&#39;organizzazione e la funzione dei cromosomi eucariotiche. Due insiemi di ciascuna di tali istoni formano la ottamero istoni, un rocchetto molecolare che dirige l&#39;avvolgimento ~ 147 paia di basi del DNA su se stessa, che si traduce nella formazione di un nucleosoma 1. I nucleosomi sono partecipanti attivi in ​​una varietà di processi cromosomiche-based, come ad esempio la regolazione della trascrizione genica e la formazione di eucromatina e heterochromatin tra cromosomi, e come tali sono stati al centro di un&#39;intensa ricerca nel corso degli ultimi decenni. Un certo numero di meccanismi sono stati descritti da cui nucleosomi può essere manipolato in modi che possono facilitare l&#39;esecuzione di processi specifici - Questi meccanismi comprendono modificazione post-traslazionale di residui degli istoni, rimodellamento nucleosomi ATP-dipendente, e la riorganizzazione nucleosomi ATP-indipendentee montaggio / smontaggio 2, 3. Il lievito Saccharomyces cerevisiae erba è particolarmente potente organismo modello per la comprensione della funzione istone negli eucarioti. Questo può essere in gran parte attribuito all&#39;elevato grado di conservazione evolutiva delle proteine istone tutto il eucarioti dominio e la riconducibilità di lievito ad una varietà di sperimentale genetico e biochimico approcci 4. approcci Reverse-genetici nel lievito sono stati ampiamente utilizzati per studiare gli effetti di specifiche mutazioni istoni su vari aspetti della biologia della cromatina. Per questi tipi di esperimenti è spesso preferibile utilizzare cellule in cui gli istoni mutanti sono espressi dal loro loci genomici nativa, come espressione da plasmidi autonome può portare a anormali livelli intracellulari di proteine ​​istoni (a causa di un numero variabile di plasmidi in cellule) e alterazione concomitante di cromatina environments, che alla fine possono confondere l&#39;interpretazione dei risultati. Qui, si descrive una tecnica PCR-based che consente di mutagenesi mirata di geni istone alle loro posizioni nativi genomiche che non richiede una fase di clonazione e risultati nella generazione della mutazione desiderata (s) senza rimanenti sequenze di DNA esogene nel genoma. Questa tecnica sfrutta l&#39;efficiente sistema di ricombinazione omologa nel lievito e ha diverse caratteristiche in comune con altre tecniche simili sviluppati da altri gruppi - in particolare il Delitto Perfetto, genomica mutagenesi sito-specifica (SSG), e la clonazione senza PCR allele-based metodi sostitutivi 5, 6, 7. Tuttavia, la tecnica descriviamo ha un aspetto che lo rende particolarmente adatto per la mutagenesi dei geni istoni. In cellule di lievito aploidi, ciascuno dei quattro istoni nucleo è codificata da due non-ageni llelic e altamente omologhi: per esempio, l&#39;istone H3 vengono codificati dai geni HHT1 e HHT2, e le fasi di lettura aperte (ORF) dei due geni sono oltre il 90% identica in sequenza. Questo elevato grado di omologia può complicare esperimenti progettati per indirizzare specificamente uno dei due geni istone-codifica per la mutagenesi. Considerando che i suddetti metodi richiedono spesso l&#39;uso di almeno alcune sequenze all&#39;interno della ORF del gene bersaglio di guidare ricombinazione omologa, la tecnica descriviamo qui fa uso di sequenze fiancheggianti le ORF dei geni istoni (che condividono molto meno omologia di sequenza) per la fase di ricombinazione, aumentando così la probabilità di successo di targeting mutagenesi al locus desiderata. Inoltre, le regioni omologhe che guidano la ricombinazione possono essere molto ampia, contribuendo ulteriormente alla efficiente ricombinazione omologa mirato.

<table><tbody><tr><td>1 kb DNA Ladder (DNA standards)</td><td>New England BioLabs</td><td>N3232L</td><td></td></tr><tr><td>Agarose&amp;nbsp;</td><td>Sigma</td><td>A5093-100G</td><td></td></tr><tr><td>Boric Acid</td><td>Sigma</td><td>B0394-500G</td><td></td></tr><tr><td>dNTP mix (10 mM each)</td><td>ThermoFisher Scientific</td><td>R0192</td><td></td></tr><tr><td>EDTA solution (0.5 M, pH 8.0)</td><td>AmericanBio</td><td>AB00502-01000</td><td></td></tr><tr><td>Ethanol (200 Proof)</td><td>Fisher Scientific</td><td>16-100-824</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA)</td><td>Sigma</td><td>E4884-500G</td><td></td></tr><tr><td>Lithium acetate dihydrate</td><td>Sigma</td><td>L6883-250G</td><td></td></tr><tr><td>MyCycler Thermal Cycler</td><td>BioRad</td><td>170-9703</td><td></td></tr><tr><td>Poly(ethylene glycol) (PEG)</td><td>Sigma</td><td>P3640-1KG</td><td></td></tr><tr><td>PrimeSTAR HS DNA Polymerase (high fidelity DNA polymerase)&amp;nbsp; and 5x buffer</td><td>Fisher Scientific</td><td>50-443-960</td><td></td></tr><tr><td>Salmon sperm DNA solution</td><td>ThermoFisher Scientific</td><td>15632-011</td><td></td></tr><tr><td>Sigma 7-9 (Tris base, powder form)</td><td>Sigma</td><td>T1378-1KG</td><td></td></tr><tr><td>Sodium acetate trihydrate</td><td>Sigma</td><td>236500-500G</td><td></td></tr><tr><td>Supra Sieve GPG Agarose (low metling temperature agarose)</td><td>AmericanBio</td><td>AB00985-00100</td><td></td></tr><tr><td>Taq Polymerase and 10x Buffer</td><td>New England BioLabs</td><td>M0273X</td><td></td></tr><tr><td>Toothpicks</td><td>Fisher Scientific</td><td>S67859</td><td></td></tr><tr><td>Tris-HCl (1 M, pH 8.0)</td><td>AmericanBio</td><td>AB14043-01000</td><td></td></tr><tr><td>a-D(+)-Glucose</td><td>Fisher Scientific</td><td>AC170080025</td><td>for yeast media</td></tr><tr><td>Agar</td><td>Fisher Scientific</td><td>DF0140-01-0</td><td>for yeast media</td></tr><tr><td>Peptone</td><td>Fisher Scientific</td><td>DF0118-07-2</td><td>for YPD medium</td></tr><tr><td>Yeast Extract</td><td>Fisher Scientific</td><td>DF0127-17-9</td><td>for YPD medium</td></tr><tr><td>4-aminobenzoic acid</td><td>Sigma</td><td>A9878-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>Adenine</td><td>Sigma</td><td>A8626-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>Glycine hydrochloride</td><td>Sigma</td><td>G2879-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Alanine</td><td>Sigma</td><td>A7627-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Arginine monohydrochloride</td><td>Sigma</td><td>A5131-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Asparagine monohydrate</td><td>Sigma</td><td>A8381-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Aspartic acid sodium salt monohydrate</td><td>Sigma</td><td>A6683-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Cysteine hydrochloride monohydrate</td><td>Sigma</td><td>C7880-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Glutamic acid hydrochloride</td><td>Sigma</td><td>G2128-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Glutamine</td><td>Sigma</td><td>G3126-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Histidine monohydrochloride monohydrate</td><td>Sigma</td><td>H8125-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Isoleucine</td><td>Sigma</td><td>I2752-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Leucine</td><td>Sigma</td><td>L8000-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Lysine monohydrochloride</td><td>Sigma</td><td>L5626-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Methionine</td><td>Sigma</td><td>M9625-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Phenylalanine</td><td>Sigma</td><td>P2126-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Proline</td><td>Sigma</td><td>P0380-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Serine</td><td>Sigma</td><td>S4500-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Threonine</td><td>Sigma</td><td>T8625-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Tryptophan</td><td>Sigma</td><td>T0254-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Tyrosine</td><td>Sigma</td><td>T3754-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>L-Valine</td><td>Sigma</td><td>V0500-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>myo-Inositol</td><td>Sigma</td><td>I5125-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>Uracil</td><td>Sigma</td><td>U0750-100G</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>Ammonium Sulfate</td><td>Fisher Scientific</td><td>A702-500</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>Yeast Nitrogen Base</td><td>Fisher Scientific</td><td>DF0919-07-3</td><td>for complete minimal dropout medium&amp;nbsp;</td></tr><tr><td>5-Fluoroorotic acid (5-FOA)</td><td>AmericanBio</td><td>AB04067-00005</td><td>for&amp;nbsp; 5-FOA medium</td></tr></tbody></table>

targeted in situ mutagenesis of histone genes in budding yeast

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting mutant alleles reside at their endogenous genomic sites and no exogenous DNA sequences are left in the genome following the procedure. Since in haploid yeast cells each of the four core histone proteins is encoded by two non-allelic genes with highly homologous open reading frames (ORFs), targeting mutagenesis specifically to one of two genes encoding a particular histone protein can be problematic. The strategy we describe here bypasses this problem by utilizing sequences outside, rather than within, the ORF of the target genes for the homologous recombination step. Another feature of this system is that the regions of DNA driving the homologous recombination steps can be made to be very extensive, thus increasing the likelihood of successful integration events. These features make this strategy particularly well-suited for histone gene mutagenesis, but can also be adapted for mutagenesis of other genes in the yeast genome.

Descriviamo la generazione di un allele hht2 esprimere un istone H3 mutante proteina ospitare una sostituzione nella posizione 53 da un arginina ad acido glutammico (H3-R53E mutante) come un esempio rappresentativo della mirata strategia situ mutagenesi. Abbiamo generato un ceppo in cui l&#39;intera ORF di HHT2 è sostituito dal gene URA3 (vedi punto 1 del protocollo). Questo ceppo,...

Watch this Scientific Journal Video about Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast at JoVE.com

Mirato alla mutagenesi in situ dei geni istonici nel lievito in erba | Genetica | Video

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

A strategy for generating mutations in histone genes at their endogenous location in Saccharomyces cerevisiae is presented.

Mirata<em&gt; In Situ</em&gt; mutagenesi dell&#39;istone geni in germogliamento lievito

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

targeted-in-situ-mutagenesis-of-histone-genes-in-budding-yeast

Universidad San Sebastian

Research

Education

JoVE Journal

JoVE Core

Biology

Genetics

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

DNA Structure and Function

SCIENTISTS IN ACTION

15.2K Views.  Hendrix College. A strategy for generating mutations in histone genes at their endogenous location in Saccharomyces cerevisiae is presented. 

Video: Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

Optogenetic caso Mutagenesi Utilizzando istone-miniSOG in<em&gt; C. elegans</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Ricerca

Genetica

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes that add or remove these marks in response to signals received by the cell. These PTMS are key contributors to the regulation of processes such as gene expression control and DNA repair. Chromatin immunoprecipitation (chIP) has been an instrumental approach for dissecting the abundance and localization of many histone PTMs throughout the genome in response to diverse perturbations to the cell. Here, a versatile method for performing chIP of post-translationally modified histones from the budding yeast Saccharomyces cerevisiae (S. cerevisiae) is described. This method relies on crosslinking of proteins and DNA using formaldehyde treatment of yeast cultures, generation of yeast lysates by bead beating, solubilization of chromatin fragments by micrococcal nuclease, and immunoprecipitation of histone-DNA complexes. DNA associated with the histone mark of interest is purified and subjected to quantitative PCR analysis to evaluate its enrichment at multiple loci throughout the genome. Representative experiments probing the localization of the histone marks H3K4me2 and H4K16ac in wildtype and mutant yeast are discussed to demonstrate data analysis and interpretation. This method is suitable for a variety of histone PTMs and can be performed with different mutant strains or in the presence of diverse environmental stresses, making it an excellent tool for investigating changes in chromatin dynamics under different conditions.

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

Here, we describe a protocol for chromatin immunoprecipitation of modified histones from the budding yeast Saccharomyces cerevisiae. Immunoprecipitated DNA is subsequently used for quantitative PCR to interrogate the abundance and localization of histone post-translational modifications throughout the genome.

Immunoprecipitazione della cromatina (ChIP) di modificazioni istoniche da Saccharomyces cerevisiae

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes ...

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve random mutagenesis, error-prone PCR is a convenient and efficient strategy for generating a diverse pool of mutants (i.e., a mutant library). Error-prone PCR is the method of choice when a researcher seeks to mutate a pre-defined region, such as the coding region of a gene while leaving other genomic regions unaffected. After the mutant library is amplified by error-prone PCR, it must be cloned into a suitable plasmid. The size of the library generated by error-prone PCR is constrained by the efficiency of the cloning step. However, in the fission yeast, Schizosaccharomyces pombe, the cloning step can be replaced by the use of a highly efficient one-step fusion PCR to generate constructs for transformation. Mutants of desired phenotypes may then be selected using appropriate reporters. Here, we describe this strategy in detail, taking as an example, a reporter inserted at centromeric heterochromatin.

This article describes a detailed methodology for random mutagenesis of a target gene in fission yeast. As an example, we target rpt4+, which encodes a subunit of the 19S proteasome, and screen for mutations that destabilize heterochromatin.

Gene targeting mutagenesi casuale per selezionare eterocromatina-destabilizzare Proteasome mutanti in lievito di fissione

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve ...

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene&#160;NDC80&#160;during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in&#160;other yeast strains and growth conditions.

Single Molecule Fluorescence In Situ Hybridization smFISH Analysis in Budding Yeast Vegetative Growth and Meiosis

This single molecule fluorescence in situ hybridization protocol is optimized to quantify the number of RNA molecules in budding yeast during vegetative growth and meiosis.

Singola molecola l'ibridazione In Situ (smFISH) analisi in crescita vegetativa lievito germogliante e meiosi

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

Alterazioni di modificazione post-traduzionale istone caratterizzante nel lievito Neurodegenerative Proteinopathy modelli

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely related biochemical pathways. Previous methods such as the Synthetic Genetic Array (SGA) analysis, and random mutagenesis techniques using ultraviolet (UV) or chemicals like ethyl methanesulfonate (EMS) or N-ethyl-N- nitrosourea (ENU), have been widely used but are often costly and laborious. Also, these mutagen-based screening methods are frequently associated with severe side effects on the organism, inducing multiple mutations that add to the complexity of isolating the suppressors. Here, we present a simple and effective protocol to identify suppressor mutations in mutants which confer a growth defect in Schizosaccharomyces pombe. The fitness of cells with a growth deficiency in standard rich liquid media or synthetic liquid media can be monitored for recovery using an automated 96-well plate reader over an extended period. Once a cell acquires a suppressor mutation in the culture, its descendants outcompete those of the parental cells. The recovered cells that have a competitive growth advantage over the parental cells can then be isolated and backcrossed with the parental cells. The suppressor mutations are then identified using whole-genome sequencing. Using this approach, we have successfully isolated multiple suppressors that alleviate the severe growth defects caused by loss of Elf1, an AAA+ family ATPase that is important in nuclear mRNA transport and maintenance of genomic stability. There are currently over 400 genes in S. pombe with mutants conferring a growth defect. As many of these genes are uncharacterized, we propose that our method will hasten the identification of novel functional interactions with this user-friendly, high-throughput approach.

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

We present a simple suppressor screen protocol in fission yeast. This method is efficient, mutagen-free, and&#160; selective&#160; for&#160; mutations that&#160; often&#160; occur at&#160; &#160;a&#160; single&#160; genomic&#160; locus.&#160; The protocol is suitable for isolating suppressors that alleviate growth defects in liquid culture that are caused by a mutation or a drug.

Una schermata di soppressore Deep-sequenziamento-assistita, spontanea nel lievito a fissione Schizosaccharomyces pombe

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely ...

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

Studi genetici di proteine ​​umane di riparazione del DNA Utilizzando lievito come sistema modello

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Biologia

Isolamento della cromatina locus-specifica da cellule di lievito

Single-Copy Gene Locus Chromatin Purification in Saccharomyces cerevisiae

The basic organizational unit of eukaryotic chromatin is the nucleosome core particle (NCP), which comprises DNA wrapped ~1.7 times around a histone octamer. Chromatin is defined as the entity of NCPs and numerous other protein complexes, including transcription factors, chromatin remodeling and modifying enzymes. It is still unclear how these protein-DNA interactions are orchestrated at the level of specific genomic loci during different stages of the cell cycle. This is mainly due to the current technical limitations, which make it challenging to obtain precise measurements of such dynamic interactions. Here, we describe an improved method combining site-specific recombination with an efficient single-step affinity purification protocol to isolate a single-copy gene locus of interest in its native chromatin state. The method allows for the robust enrichment of the target locus over genomic chromatin, making this technique an effective strategy for identifying and quantifying protein interactions in an unbiased and systematic manner, for example by mass spectrometry. Further to such compositional analyses, native chromatin purified by this method likely reflects the in vivo situation regarding nucleosome positioning and histone modifications and is, therefore, amenable to further structural and biochemical analyses of chromatin derived from virtually any genomic locus in yeast.

Autore in primo piano: Avanzamento della ricerca sulla cromatina e superamento dei limiti con un protocollo di isolamento della cromatina specifico per locus ad alto arricchimento 

This protocol presents a locus-specific chromatin isolation method based on site-specific recombination to purify a single-copy gene locus of interest in its native chromatin context from budding yeast, Saccharomyces cerevisiae.

Purificazione della cromatina del locus genico a copia singola in Saccharomyces cerevisiae

The basic organizational unit of eukaryotic chromatin is the nucleosome core particle (NCP), which comprises DNA wrapped ~1.7 times around a histone ...

Mirata

Riepilogo

Esplora Altri Video

Capitoli in questo video

Optogenetic caso Mutagenesi Utilizzando istone-miniSOG in

Immunoprecipitazione della cromatina (ChIP) di modificazioni istoniche da Saccharomyces cerevisiae

Gene targeting mutagenesi casuale per selezionare eterocromatina-destabilizzare Proteasome mutanti in lievito di fissione

Singola molecola l'ibridazione In Situ (smFISH) analisi in crescita vegetativa lievito germogliante e meiosi

Alterazioni di modificazione post-traduzionale istone caratterizzante nel lievito Neurodegenerative Proteinopathy modelli

Una schermata di soppressore Deep-sequenziamento-assistita, spontanea nel lievito a fissione Schizosaccharomyces pombe

Studi genetici di proteine umane di riparazione del DNA Utilizzando lievito come sistema modello

Purificazione della cromatina del locus genico a copia singola in Saccharomyces cerevisiae

Purificazione della cromatina del locus genico a copia singola in Saccharomyces cerevisiae

Purificazione della cromatina del locus genico a copia singola in Saccharomyces cerevisiae

Mirata

Riepilogo

Esplora Altri Video

Capitoli in questo video

Optogenetic caso Mutagenesi Utilizzando istone-miniSOG in

Immunoprecipitazione della cromatina (ChIP) di modificazioni istoniche da Saccharomyces cerevisiae

Gene targeting mutagenesi casuale per selezionare eterocromatina-destabilizzare Proteasome mutanti in lievito di fissione

Singola molecola l'ibridazione In Situ (smFISH) analisi in crescita vegetativa lievito germogliante e meiosi

Alterazioni di modificazione post-traduzionale istone caratterizzante nel lievito Neurodegenerative Proteinopathy modelli

Una schermata di soppressore Deep-sequenziamento-assistita, spontanea nel lievito a fissione Schizosaccharomyces pombe

Studi genetici di proteine ​​umane di riparazione del DNA Utilizzando lievito come sistema modello

Purificazione della cromatina del locus genico a copia singola in Saccharomyces cerevisiae

Purificazione della cromatina del locus genico a copia singola in Saccharomyces cerevisiae

Purificazione della cromatina del locus genico a copia singola in Saccharomyces cerevisiae

Studi genetici di proteine umane di riparazione del DNA Utilizzando lievito come sistema modello